Compound having photocleavage activity or salt thereof

Abstract

To provide a compound having improved photocleavage activity and improved hydrolysis resistance.SOLUTION: A compound in which a silyl group represented by the following formula is introduced into a β - position of a carbocation of a photoexcited species: wherein R1a, R1b, and R1c are each independently an arbitrary hydrocarbyl group.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a compound having photocleavage activity or a salt thereof, a composition containing the compound or a salt thereof, a method for obtaining the compound or a salt thereof by light irradiation, a method for improving the photocleavage activity of a compound having photocleavage activity or a salt thereof, and a method for improving the hydrolysis resistance of a compound having photocleavage activity or a salt thereof. [Background technology]

[0002] Photocleavable compounds have the property of absorbing light of a specific wavelength and using the energy to cleave chemical bonds within a molecule. Applications of photocleavable compounds have been explored in various fields. For example, if a physiologically active molecule is modified with a protecting group that can be removed by light irradiation to suppress the physiological activity, temporal and spatial control of the physiological activity by light irradiation becomes possible. Furthermore, if a molecule is bound to a carrier via a photocleavable group, the molecule can be released from the carrier by light irradiation. For this reason, photocleavable compounds have been explored for applications as molecular tools for understanding biological functions and as stimuli-responsive materials (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Wegner, S., Sentuerk, O. & Spatz, J. "Photocleavable linker for the patterning of bioactive molecules." Sci Rep 5, 18309 (2016). https: / / doi.org / 10.1038 / srep18309 Summary of the Invention [Problem to be solved by the invention]

[0004] As described above, compounds with photocleavage activity absorb light of a specific wavelength and use the light energy to cleave chemical bonds within molecules. Because ultraviolet light is cytotoxic and DNA-damaging, compounds with photocleavage activity used in fields such as biology and medicine are preferably cleavable by visible light. However, compounds that can be cleaved by visible light have problems such as particularly low photocleavage activity and low stability against hydrolysis (i.e., hydrolysis resistance). Furthermore, there is still room for improvement in the photocleavage activity and hydrolysis resistance of compounds that can be cleaved by light other than visible light.

[0005] As mentioned above, compounds with photocleavage activity are being considered for application in technologies that temporally and spatially control molecular activity and behavior depending on whether or not light is irradiated. However, if the photocleavage activity of the compound is low, the expected activity and behavior may not be exhibited even when irradiated with light. Furthermore, if the compound's resistance to hydrolysis is low, the bond may be cleaved by hydrolysis even without light irradiation, and activity may be exhibited at unexpected times and places.

[0006] Therefore, improving the photocleavage activity and hydrolysis resistance of a compound having photocleavage activity is important from the viewpoint of improving the precision of techniques for temporally and spatially controlling the activity, behavior, etc. of molecules. Therefore, the present inventors set a primary objective to provide a compound having photocleavage activity with improved photocleavage activity and improved hydrolysis resistance. [Means for solving the problem]

[0007] The present inventors have found that a compound of the following formula: [ka] (In the formula, R 1a , R 1b , and R 1c are each independently any hydrocarbon group. It has been found that by introducing a silyl group represented by the formula (I), the photoexcited species can be chemically stabilized, and as a result, the photocleavage activity of the compound or its salt can be improved. It has also been found that the hydrophobicity and bulkiness of the hydrocarbon group contained in the silyl group can improve the hydrolysis resistance of the compound or its salt. Further improvements have been made, and the present disclosure has been completed.

[0008] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. General formula (I): [ka] (In the formula, R 1a , R 1b , and R 1c are each independently any hydrocarbon group; R 2 is any group containing a fluorescent moiety; X is NH or CH; Y 1 is any group) or a salt thereof, Upon irradiation with light, the compound of general formula (II): [ka] (In the formula, R 1a , R 1b , R 1c , and R 2 is the same as in general formula (I). A composition for producing a compound represented by the formula: Section 2. In general formula (I), the R 2 is represented by the general formula (III): [ka] (In the formula, R 3 is any electron donating group; R 4a , R 4b , R 4c , and R 4d are each independently a hydrogen atom or any group. The composition according to item 1, represented by: Section 3. In general formula (I), R 1a , R 1b , and R 1c are each independently an alkyl group having 1 to 5 carbon atoms or a phenyl group. Section 4. Item 4. The composition according to any one of Items 1 to 3, wherein the phosphor portion absorbs light having a wavelength of 350 nm to 850 nm, and the light irradiation is light having a wavelength of 350 nm to 850 nm. Section 5. General formula (I): [ka] (In the formula, R 1 are each independently any hydrocarbon group; R 2 is any group containing a fluorescent moiety; X is NH or CH; Y 1 is any group) or a salt thereof, General formula (II): [ka] (In the formula, R 1a , R 1b , R 1c , and R 2 is the same as in general formula (I). A method for obtaining a compound represented by the formula: Section 6. General formula (IV): [ka] (In the formula, R 1a , R 1b , and R 1c are each independently any hydrocarbon group; R 3 is any electron donating group; X is NH or CH; Y 1 is any group) A compound or a salt thereof represented by the formula: [Effects of the Invention]

[0009] According to the present disclosure, a compound or a salt thereof having photocleavage activity, which has improved photocleavage activity and improved hydrolysis resistance, is provided. Furthermore, according to the present disclosure, there are provided a method for obtaining a compound or a salt thereof by light irradiation, a method for improving the photocleavage activity of a compound or a salt thereof having photocleavage activity, a method for improving the hydrolysis resistance of a compound or a salt thereof having photocleavage activity, etc. [Brief explanation of the drawings]

[0010] [Figure 1a] 1 shows a synthesis scheme for the compound described in Test 1. [Figure 1b] (Continuation of Figure 1a) The synthesis scheme of the compound described in Test 1 is shown. [Figure 2] The compounds described in Test 2 are shown. [Figure 3a] The results of Test 3 are shown in Figure 3a. The upper part of Figure 3a shows the reaction of compound 11 being cleaved by light irradiation. The lower part of Figure 3a shows the change in the 1H NMR signal of compound 11 over time due to light irradiation. [Figure 3b] The results of Test 3 are shown in Figure 3b. The upper part of Figure 3b shows the reaction of compounds cleaved by light irradiation. The lower part of Figure 3b shows the photocleavage rate when compounds 9 to 13 were each irradiated with light at 14 mW for 30 minutes. [Figure 4] A schematic diagram of the (8a")-loaded magnetic beads prepared in Test 4 is shown. The upper part of Figure 4 shows how the portion containing HaloTag-fused NanoLuc is separated from the (8a")-loaded magnetic beads by photocleavage or hydrolysis. The lower part of Figure 4 shows the state (enlarged view) of compound (8a") immobilized on the magnetic bead solid phase. [Figure 5]The results of Test 5 are shown below. The vertical axis on the left side of Figure 5 represents luminescence intensity, and the horizontal axis represents light irradiation time (seconds). The center of Figure 5 shows the structures of the parts involved in the photocleavage and hydrolysis of compounds (6a”, 6b”, 8a”, 8b”) (the structures of other parts are omitted). The right side of Figure 5 shows the luminescence intensity of each evaluation sample after 3 minutes of light irradiation. [Figure 6] The results of Test 6-1 are shown. The vertical axis of each graph indicates luminescence intensity. [Figure 7] The graph shows the results of Test 6-2. The vertical axis of the graph indicates luminescence intensity. DETAILED DESCRIPTION OF THE INVENTION

[0011] Each embodiment included in the present disclosure will be described in more detail below. The present disclosure preferably includes, but is not limited to, a compound having photocleavage activity or a salt thereof, a composition containing the compound or a salt thereof, a method for obtaining a compound or a salt thereof by light irradiation, a method for improving the photocleavage activity of a compound having photocleavage activity or a salt thereof, and a method for improving the hydrolysis resistance of a compound having photocleavage activity or a salt thereof. The present disclosure includes all that is disclosed herein and that can be recognized by a person skilled in the art. Hereinafter, when simply referring to a "compound" in the present disclosure, unless otherwise specified, it includes not only the compound itself but also any salt that the compound may take.

[0012] 1. Compounds or salts thereof having photocleavage activity Compounds having photocleavage activity encompassed by the present disclosure have the following general formula (I): [ka] (In the formula, R 1a , R 1b , and R 1c are each independently any hydrocarbon group; R 2 is any group containing a fluorescent moiety; X is NH or CH; Y 1 is any group). Hereinafter, this compound may be referred to as the "compound of the present disclosure."

[0013] In general formula (I), R 1a , R 1b , and R 1c are each independently any hydrocarbon group. The specific structure of the hydrocarbon group is not particularly limited, and may be, for example, an alkyl group, an alkenyl group, an alkynyl group, an aromatic hydrocarbon group, or the like. The alkyl group, alkenyl group, and alkynyl group may be linear or branched, or may be cyclic. The hydrocarbon group may also be a group having a structure in which the hydrocarbon group is further substituted with another hydrocarbon group, or a group having a structure in which these groups are linked together. The number of carbon atoms constituting the hydrocarbon group is not particularly limited, and may be, for example, 1 to 18 carbon atoms, or 1 to 12 carbon atoms, preferably 1 to 6, and particularly preferably 1 to 3.

[0014] Specific examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a hexyl group, a heptyl group, an octyl group, a 2-ethylhexyl group, a nonyl group, a decyl group, a dodecyl group, a tetradecyl group, a pentadecyl group, an octadecyl group, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, and a cyclooctyl group.

[0015] Specific examples of the alkenyl group include a vinyl group, a 1-propenyl group, a 2-propenyl group, an isopropenyl group, a 1-butenyl group, a 2-butenyl group, a 3-butenyl group, a 1-octenyl group, a 1-decenyl group, and a 1-octadecenyl group.

[0016] Specific examples of the alkynyl group include an ethynyl group, a 1-propynyl group, a 2-propynyl group, a 1-butynyl group, a 2-butynyl group, a 3-butynyl group, a 1-octynyl group, a 1-decynyl group, and a 1-octadecynyl group.

[0017] Specific examples of aromatic hydrocarbon groups include groups obtained by removing one hydrogen atom from benzene, toluene, xylene, mesitylene, naphthalene, or anthracene. From the viewpoint of ease of chemical synthesis, among aromatic hydrocarbon groups, groups obtained by removing one hydrogen atom from benzene or toluene (i.e., phenyl group or benzyl group) are preferred, and phenyl group is particularly preferred.

[0018] From the viewpoint of ease of chemical synthesis, in general formula (I), R 1a , R 1b , and R 1c are each independently preferably an alkyl group having 1 to 5 carbon atoms, or a group obtained by removing one hydrogen atom from benzene or toluene (i.e., a phenyl group or a benzyl group), more preferably an alkyl group having 1 to 3 carbon atoms or a phenyl group, and particularly preferably a methyl group, an ethyl group, or a phenyl group.

[0019] In general formula (I), R 2 is any group containing a fluorescent moiety. In the present disclosure, the term "fluorophore moiety" refers to a molecular structure capable of absorbing light energy to enter an excited state and then emitting light energy (i.e., emitting fluorescence). It is generally known that molecules capable of emitting fluorescence have conjugated double bonds.

[0020] Specific examples of the fluorescent moiety include molecular skeletons known as fluorescent dyes, such as a coumarin skeleton, a cyanine skeleton, a BODIPY (boron-dipyrromethene) skeleton, a xanthene skeleton, a pyrene skeleton, and a squarium skeleton. The structures of the coumarin skeleton, the cyanine skeleton, the BODIPY skeleton, and the xanthene skeleton are shown below. [ka] [ka] (In the formula, m is any natural number, and may be, for example, 1 to 10, 1 to 5, or 1 to 4.) [ka] [ka]

[0021] Also, the following formula: [ka] The skeleton structure represented by the following formula is also a preferred example of the structure of the phosphor moiety.

[0022] R 2 may be the above-mentioned phosphor moiety itself, or may be a phosphor moiety to which an arbitrary group is added. 2 comprises at least one fluorescent moiety selected from the group consisting of a coumarin skeleton, a cyanine skeleton, and a BODIPY skeleton, and more preferably R 2 contains a coumarin skeleton. 2 As an embodiment of the present invention, for example, the general formula (III): [ka] (In the formula, R 3 is any electron-donating group; R 4a , R 4b , R 4c , and R 4d are each independently a hydrogen atom or any group).

[0023] In general formula (III), R 3 is any electron-donating group. It is known that the addition of an electron-donating group to a coumarin skeleton improves the light absorption ability of the coumarin skeleton. Specific groups that have electron-donating properties are known in the art. For example, alkyl groups, alkoxy groups, and -N-(R 5 )2(R 5are each independently a hydrogen atom or an arbitrary hydrocarbon group), and the like. Here, the number of carbon atoms in the alkyl group, alkoxy group, and hydrocarbon group is not particularly limited, and may be, for example, 1 to 18, 1 to 12, 1 to 8, 1 to 6, or 1 to 3. 5 Examples of the electron-donating group include a hydrogen atom, a methyl group, an ethyl group, and a propyl group. [ka] and the following formula: [ka] Preferred examples include groups represented by the following formula: R 3 These electron donating groups may further include any group (e.g., Y 2 ) may be added.

[0024] Preferred R 3 Specific embodiments of the present invention are as follows: [ka] (In the formula, Y 2 is a hydrogen atom or any group. n is any natural number, for example, it may be 1 to 18, 1 to 12, 1 to 8, 1 to 6, or 1 to 3.

[0025] In general formula (III), R 4a , R 4b , R 4c , and R 4d are each independently a hydrogen atom or any group. 4a , R 4b , R 4c , and R 4d When at least one of the groups is an arbitrary group, the structure of the group is not particularly limited as long as the effects of the present disclosure are achieved. For example, the group may be a hydrocarbon group or an alkoxy group having about 1 to 6 carbon atoms, or may be a halogen atom. Furthermore, the group may form a ring structure together with the carbon atoms constituting the coumarin skeleton.

[0026] Y 2 Y is a hydrogen atom or any group, and its specific structure is not particularly limited as long as the effects of the present disclosure are achieved. 2 is located away from the site of photocleavage, so Y 2 The structure of Y is believed to have little effect on the photocleavage activity and hydrolysis resistance of the compounds of the present disclosure. 2 may have a structure such as a nucleic acid, polypeptide, or protein, may have a structure for binding to a carrier, or may have a structure capable of exerting physiological activity. In addition to these, it may also have a linker structure such as an alkyl linker, a polyalkylene glycol linker, or a polyamide linker.

[0027] In general formula (I), R 2 There is no particular limitation on the molecular weight of R. 2 If is the phosphor part itself, R 2 The molecular weight of R is about 100 to several hundred. 2 Y 2 and the Y 2 If is an antibody, then R 2 It is expected that the molecular weight of Y may exceed one million. 2 The structure of R is thought to have little effect on the photocleavage activity and hydrolysis resistance of the compounds of the present disclosure. 2 It is reasonably expected that the effects of the present disclosure will be similarly obtained whether the molecular weight of R is, for example, about 100 or exceeds one million. 2 The molecular weight may be, for example, about 100 to 5,000,000, or about 140 to 2,000,000. The upper or lower limit of the range may be, for example, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 500,000, 1,000,000, or 5,000,000.

[0028] In general formula (I), X is NH or CH. When X is NH, the compound of the present disclosure undergoes a photocleavage reaction to produce the following compound: [ka] When X is CH2, the compounds of the present disclosure undergo photocleavage to give the following compounds: [ka]

[0029] In general formula (I), Y 1 is any group. Y 1 The specific structure of the above is not particularly limited as long as the effects of the present disclosure are achieved. 2 Similarly, Y 1 is located away from the site where photocleavage occurs, 1 The structure of Y is believed to have little effect on the photocleavage activity and hydrolysis resistance of the compounds of the present disclosure. 1 may be, for example, any organic group, may have a structure such as a nucleic acid, polypeptide, or protein, may have a structure for binding to any carrier, or may have a structure capable of exerting physiological activity. In addition to these, it may also have a linker structure such as an alkyl linker, a polyalkylene glycol linker, or a polyamide linker.

[0030] Preferred Y 1 Specific embodiments of the formula include, for example, an optionally substituted phenyl group and a group represented by the following formula: [ka] (In the formula, Y 3 is a hydrogen atom or any group. m is any natural number, for example, it may be 1 to 18, or it may be 1 to 12, 1 to 8, 1 to 6, or 1 to 3).

[0031] Y 3When is any group, examples of the group include a hydroxyl group, an optionally substituted linear or branched alkyl group, an optionally substituted cycloalkyl group, an optionally substituted aromatic carbocyclic group, and groups to which a nucleic acid, polypeptide, protein, etc. is linked directly or via an optional linker. The number of carbon atoms constituting the alkyl group, cycloalkyl group, aromatic carbocyclic group, etc. is not particularly limited and may be, for example, 1 to 18, 1 to 12, 1 to 8, 1 to 6, or 1 to 3.

[0032] Furthermore, the substituents in the aforementioned optionally substituted phenyl group, optionally substituted linear or branched alkyl group, optionally substituted cycloalkyl group, and optionally substituted aromatic carbocyclic group are not particularly limited, and examples thereof include halogen (particularly F, Cl, Br, or I) or linear or branched alkyl groups having 1 to 6 carbon atoms (1, 2, 3, 4, 5, or 6). The number of such substituents may be, for example, 1, 2, or 3.

[0033] Although not particularly limited, a linear or branched alkyl group having 1 to 18 carbon atoms which may be substituted with halogen is more preferred. 3 It is cited as one form of.

[0034] In general formula (I), Y 1 The molecular weight of the copolymer is not particularly limited. For example, it may be about 10 to 5,000,000, or about 15 to 2,000,000. The upper or lower limit of the range may be, for example, 20, 50, 100, 200, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, or 5,000,000.

[0035] Particularly preferred embodiments of the compounds of the present disclosure include compounds of the general formula (IV): [ka] Examples of compounds represented by R 1a , R 1b , and R1c , and R 3 , X, and Y 1 is as described above.

[0036] 2. Application of the Technology of the Present Disclosure The compounds of the present disclosure can absorb light of a specific wavelength via the fluorescent moiety, resulting in photocleavage, which, when X is NH, yields the following compound: [ka] Also, when X is CH2, the following compound is produced: [ka]

[0037] That is, whether X is NH or CH, the compound of the present disclosure can be photocleaved to give the following general formula (II): [ka] Thus, the present disclosure includes a compound represented by the general formula (I) above, and also includes a composition for producing a compound represented by the general formula (II) above upon irradiation with light. 1a , R 1b , and R 1c , and R 2 , X, and Y 1 is as described in "1. Compounds having photocleavage activity." Compounds in which X is CH2 have superior photocleavage efficiency to compounds in which X is NH. The composition may contain optional components such as a solvent, a buffer, a pH adjuster, a chelating agent, etc., as long as the effects of the present disclosure are achieved.

[0038] The wavelength of light irradiated onto the compound of the present disclosure is not particularly limited as long as it is a wavelength that can be absorbed by the compound of the present disclosure, and may be, for example, 100 to 900 nm. However, because ultraviolet light has cytotoxicity and DNA damaging properties, when the compound of the present disclosure is used in the fields of biology or medicine, the wavelength of light irradiated onto the compound of the present disclosure is preferably in the visible light region. Specifically, light with a wavelength of 350 to 850 nm is preferred, light with a wavelength of 380 to 600 nm is more preferred, light with a wavelength of 390 to 500 nm is even more preferred, and light with a wavelength of 390 to 450 nm is particularly preferred.

[0039] The wavelength absorbed by the phosphor moiety of the compound of the present disclosure can be easily adjusted by appropriately selecting the skeletal structure of the phosphor moiety and the structure, number, and position of groups added to the phosphor moiety, etc. In other words, a person skilled in the art can easily obtain a compound of the present disclosure having absorption at a desired wavelength based on known techniques in the technical field and the disclosures of this specification.

[0040] The photocleavable compounds of the present disclosure are expected to be applied to technologies that can control the activity and behavior of molecules in time and space depending on the presence or absence of light irradiation. For example, R 2 and Y 1 It is envisioned that a portion having a physiological activity is introduced into one of the two, and a portion suppressing said physiological activity is introduced into the other. This makes it possible to control said physiological activity temporally and spatially by light irradiation. In another embodiment, R 2 and Y 1 It is envisioned that one of the two will be bound to an antibody with high specificity for a particular cell type (e.g., cancer cells), and the other will be bound to a drug (e.g., a drug with antitumor activity). This will allow the drug to be selectively delivered to the target cells, and then released by light irradiation.

[0041] In another embodiment, R 2 and Y 1It is envisioned that a hydrophilic structure is introduced into one of the two and a hydrophobic structure is introduced into the other. As a result, the compound, which is amphiphilic and surfactant before light irradiation, loses its surfactant properties upon light irradiation. By utilizing this property, for example, micelles can be formed with the compound before light irradiation, a lipophilic active ingredient can be encapsulated inside the micelles, and the active ingredient can be released from the micelles by light irradiation at the desired timing.

[0042] In another embodiment, R 2 and Y 1 It is assumed that a molecule is bound to one of the two and a carrier is bound to the other. This allows the molecule to be released from the carrier by light irradiation at any time. More specifically, for example, R 2 and Y 1 It is envisioned that a physiologically active substance (e.g., peptide or nucleic acid) is bound to one of the two, and the other is bound to a fine wire such as a nanowire. In this case, the substance can be delivered into a target cell or into the nucleus of a cell by the wire, and then released by light irradiation.

[0043] In yet another embodiment, R 2 and Y 1 It is envisioned that a molecule with low stability is bound to one of the two, and a molecule (protecting group) that improves the stability of the molecule is bound to the other. This increases the storage stability of the molecule with low stability, and also makes it possible to use the molecule by removing the protecting group by irradiating it with light as needed.

[0044] As described above, the compounds of the present disclosure can exhibit excellent photocleavage activity and hydrolysis resistance. Therefore, the technology of the present disclosure can be suitably used in systems containing water. Furthermore, if the compounds of the present disclosure can be cleaved by irradiation with visible light, they can be suitably used in fields such as biology and medicine, where living organisms and living cells are used.

[0045] 3. Methods for Making the Compounds of the Present Disclosure The compounds of the present disclosure can be produced from known compounds or intermediates that can be easily synthesized from known compounds by methods that can be easily conceived by a person skilled in the art based on known techniques and the disclosures of this specification, etc. More specifically, for example, the compounds of the present disclosure can be produced by the following synthesis scheme. [ka]

[0046] Each compound obtained in the process of producing the compound of the present disclosure can be isolated or purified using known methods such as solvent extraction, concentration, distillation, sublimation, recrystallization, reprecipitation, chromatography, etc. Alternatively, the compound can be subjected to a subsequent reaction step in the form of a reaction mixture or a crude product.

[0047] The compounds of the present disclosure may optionally form salts.Similarly, compounds other than the compounds of the present disclosure, such as the compound represented by general formula (II), may also optionally form salts.Specific salts include, for example, salts with inorganic bases, such as aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, potassium salts, sodium salts, and zinc salts. Furthermore, for example, salts with organic bases include salts of primary, secondary, or tertiary amines, and specific examples of amines include arginine, betaine, caffeine, choline-N-N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine-N-ethylmorpholine-N-ethylpiperidine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, and tromethamine. Salts with inorganic acids include hydrochlorides, hydrobromides, sulfates, and nitrates. Furthermore, examples of salts with organic acids include acetate, benzoate, tartrate, maleate, fumarate, succinate, citrate, oxalate, p-toluenesulfonate, benzenesulfonate, methanesulfonate, and trifluoroacetate.

[0048] 4. Possible mechanism Without wishing to be bound by any theory, it is believed that the photocleavage activity of compounds in the technology of the present disclosure is improved by the following mechanism: the compound of the present disclosure absorbs light of a specific wavelength by a fluorescent moiety or the like, and electrons are excited by the light energy (photoexcitation). Photoexcitation generates a carbocation in the compound of the present disclosure, and the silyl group present at the β-position (the position of the adjacent carbon atom) of the carbocation is thought to further stabilize the carbocation. As a result, it is thought that the reaction efficiency of the cleavage reaction, which proceeds via a reaction intermediate containing the carbocation, is improved.

[0049] Furthermore, without wishing to be bound by any theory, it is presumed that the hydrolysis resistance of the compound in the technology of the present disclosure is improved by the following mechanism: In the compound of the present disclosure, a compound represented by the following formula: [ka] (In the formula, R 1a , R 1b , and R 1c are each independently any hydrocarbon group. The bulkiness of the silyl group and the hydrophobicity of the hydrocarbon group that constitutes the silyl group prevent water molecules or hydroxide ions from contacting the carbonyl group. As a result, it is believed that the hydrolysis resistance of the compound is improved.

[0050] In this specification, the term "comprising" includes "essentially consisting of" and "consisting of" in addition to "containing." Furthermore, the present disclosure encompasses all arbitrary combinations of the constituent elements described in this specification.

[0051] Furthermore, the various characteristics (properties, structures, functions, values, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter encompassed by the present disclosure. In other words, the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein. [Example]

[0052] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0053] Test 1. Synthesis of Compounds (Figure 1a and Figure 1b) The synthesis schemes are shown in Figures 1a and 1b. Hereinafter, the synthesis methods for each compound shown in Figures 1a and 1b will be specifically described. However, since Compounds 1 and 2 in Figure 1a are conventionally known compounds, the synthesis methods will be omitted.

[0054] 1-1. Compound 3: tert-butyl 4-(4-methyl-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate [ka]

[0055] Compound 2 (521 mg), tert-butyl piperazine-1-carboxylate (759 mg), Pd(dba)CHCl(34.7 mg), X-Phos (318 mg), cesium carbonate (1.60 mg), and 1,4-dioxane (11 mL) were placed in a 50 mL recovery flask under an Ar atmosphere and stirred overnight under reflux. The reaction solution was filtered through Celite and concentrated. The residue was dissolved in ethyl acetate and washed with saturated brine. The organic layer was dried over magnesium sulfate and concentrated. The crude product was purified by column chromatography (hex: EtOAc 20–40%) to give compound 3 (343 mg, 59% yield).

[0056] 1 H NMR(500 MHz, CDCl3)δ(ppm):1.48(s, 9H),2.36(d, 3H, 4J = 1.2 Hz), 3.30(t, 4H,3J =5.1 Hz),3.59(t, 4H, 3J = 5.1 Hz), 6.06(s, 1H),6.71(d, 1H, 4J = 2.5 Hz), 6.81(dd, 1H, 3J =8.9 Hz, 4J =2.5 Hz), 7.44(d, 1H, 3J = 8.9 Hz). 13HRMS(ESI, Positive-mode):m / z calcd for [C19H24N2O4+Na]+:367.1628,found 367.1632.

[0057] 1-2. Compound 4: tert-butyl 4-(4-formyl-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate [ka]

[0058] Compound 3 (343 mg) was placed in a 100 mL recovery flask and dissolved in p-xylene (10 mL). Heating was initiated. When the oil bath temperature reached 105 °C, selenium dioxide (212 mg) was added and the mixture was stirred overnight under reflux. While the reaction solution was still hot, it was filtered through Celite and concentrated. The residue was dissolved in ethyl acetate and washed with saturated brine. The organic layer was dried over magnesium sulfate and concentrated. The crude product was purified by column chromatography (Tol: EtOAc = 17:3 (v / v)) to give compound 4 (158.2 mg, 44% yield).

[0059] 1 H NMR(500 MHz, CDCl3)δ(ppm):1.48(s, 9H),3.36(t, 4H, 3J = 5.5 Hz), 3.60(t,4H, 3J =5.5 Hz),6.59(s, 1H), 6.71(d, 1H,4J =2.6 Hz), 6.84(dd,1H, 3J =9.2 Hz, 4J =2.5 Hz),8.39(d, 1H, 3J = 9.2 Hz)10.0(s,1H). 13C NMR(126 MHz)δ(ppm):28.5,43.1,47.2,80.5,101.1,106.3,112.2,120.3,127.2,134.8,153.7,154.7,156.9,161.4,192.2. HRMS(ESI,Positive-mode):m / z calcd for [C19H22N2O5+Na]+:381.1421,found 381.1426.

[0060] 1-3. Compound 5: tert-butyl 4-(4-(hydroxymethyl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate [ka]

[0061] Compound 4 (70 mg) was placed in a 50 mL recovery flask, dissolved in MeOH (10 mL), and NaBH4 (14.4 mg) was added and stirred for 20 minutes. Hydrochloric acid was added to terminate the reaction, and the reaction solution was extracted three times with ethyl acetate. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by column chromatography (DCM:MeOH 5%) to obtain compound 5 (42.0 mg, 59% yield).

[0062] 1 H NMR(500 MHz, CDCl3)δ(ppm):1.48(s,9H),3.20(t, 4H, 3J = 5.5 Hz), 3.59(t,4H,3J =5.5 Hz),4.85(d, 2H, 4J = 1.3 Hz), 6.38(t,1H,4J =1.3 Hz),6.71(d, 1H, 4J = 2.4 Hz), 6.71(dd,1H,3J =9.0 Hz,4J =2.4 Hz),7.38(d, 1H, 3J = 9.0 Hz). 13C NMR(126 MHz)δ(ppm):28.5,43.5,47.7,61.0,80.5,101.9,108.0,109.4,111.8,124.4,153.4,154.4,154.8,155.7,162.1. HRMS(ESI,Positive-mode):m / z calcd for [C19H24N2O5+Na]+:383.1577,found 383.1580.

[0063] 1-4. Compound 7: tert-butyl 4-(4-(1-hydroxy-2-(trimethylsilyl)ethyl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate [ka]

[0064] Under an Ar atmosphere, a ((trimethylsilyl)methyl)magnesium chloride THF solution (1.0 M, 1.0 mL) was added to a THF solution (10 mL) of compound 4 (225.7 mg) cooled to −78° C., and the mixture was stirred at −78° C. overnight.

[0065] The reaction was stopped by adding saturated aqueous ammonium chloride solution at room temperature. The reaction solution was extracted twice with dichloromethane. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by column chromatography to obtain compound 7 (153.3 mg, 55% yield).

[0066] 1 H NMR(500 MHz, CDCl3)δ(ppm):0.10(s,9H)1.12(m, 2H, )1.46(s,9H),3.27(t, 4H, 3J = 5.5 Hz), 3.56(t,4H,3J =5.5 Hz),5.07(m, 1H), 6.30(s,1H),6.64(br,1H),6.77(d, 1H, 3J = 8.7 Hz), 7.50(d,1H,3J =8.7 Hz). 13C NMR(126 MHz)δ(ppm):-0.7,26.4,28.5,43.0,47.6,68.4,80.4,101.9,106.5,109.1,111.6,125.3,153.0,154.7,156.0,160.9,162.5. HRMS(ESI,Positive-mode):m / z calcd for [C23H34N2O5Si+Na]+:469.2129,found 469.2138.

[0067] 1-5. Compound 6 and Compound 8 [ka]

[0068] Compound 5 or Compound 7 was dissolved in methanol under an Ar atmosphere, TMSCl was added, and the mixture was stirred at room temperature for n hours (n = 0.5 (Compound 5), 2 (Compound 7)). The solvent was then evaporated under a nitrogen flow (to give Compound 5' or Compound 7' (hydrochloride)). 4-Azidobenzoic acid was dissolved in dimethylformaldehyde and cooled to approximately 0°C. EDCl, HOBt, and NMM were added and stirred for 20 minutes. A DMF solution of Compound 5' or Compound 7' was slowly added. The mixture was then returned to room temperature and stirred for 1 hour. Ethyl acetate was added to the reaction solution, which was washed with 10% aqueous lithium chloride, ammonium chloride, and sodium bicarbonate. The organic layer was dried over magnesium sulfate and concentrated to give the target compounds in two-step yields of 63% and 83%, respectively.

[0069] Compound 6: 1 H NMR(500 MHz, CDCl3)δ(ppm):3.36(br,4H),3.75(br,4H),4.84(d, 2H, 4J = 1.3 Hz ), 6.39(t,1H,4J =1.3 Hz),6.73(d, 1H, 4J = 2.5 Hz), 6.81(dd,1H,3J =9.0 Hz,4J =2.5 Hz),7.08(pseudo-d,2H),7.39(d, 1H, 3J = 9.0 Hz), 7.46(pseudo-d, 2H). 13C NMR(126 MHz)δ(ppm):48.0,61.0,102.2,108.4,109.8,112.0,119.3,124.5,129.3,131.6,142.3,153.2,154.2,155.7,161.9,169.9. HRMS(ESI,Positive-mode):m / z calcd for [C21H19N5O4+Na]+:428.1329,found 428.1335.

[0070] Compound 8: 1 H NMR(500 MHz, CDCl3)δ(ppm):0.11(s,9H),1.14(m, 2H), 3.35(br,4H),3.78(br,4H),5.06(m, 1H, ),6.32(s, 1H,), 6.70(s,1H),6.80(d, 1H, 3J = 8.8 Hz), 7.06(pseudo-d, 2H), 7.44(pseudo-d, 2H)7.44(d,1H,3J =8.8 Hz),. 13 C NMR(126 MHz)δ(ppm):-0.7,26.5,47.9,68.4,102.2,106.9,109.5,111.8,119.3,125.4,129.3,131.6,142.3,152.8,156.1,160.7,162.3,169.9. HRMS(ESI,Positive-mode):m / z calcd for [C25H29N5O4Si+Na]+:514.1881,found 514.1890.

[0071] 1-6. Compound 6a and Compound 8a [ka]

[0072] Compound 6 or compound 8 was dissolved in a solvent, and EDCl, DMAP, and halotag ligand carboxylic acid were added and stirred for 2 hours and 30 minutes. Ethyl acetate was added to the reaction solution, and the mixture was washed with saturated brine. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by PTLC to give compounds 6a and 8a in yields of 77% and 55%, respectively.

[0073] Compound 6a: 1 H NMR(500 MHz, CDCl3)δ(ppm):. 13 C NMR(126 MHz)δ(ppm):HRMS(ESI,Positive-mode):m / z calcd for [C21H19N5O4+Na]+:733.2737,found 733.2723.

[0074] Compound 8a: 1 H NMR(500 MHz, CDCl3)δ(ppm):0.06(s,9H),1.21 - 1.30(m,4H),1.32 - 1.38(m,2H),1.40 - 1.46(m,2H),3.​​​​​​​​​​​​​​​Compound 6 or compound 8 was dissolved in a solvent, diisopropylethylamine was added, and the mixture was stirred at room temperature for 10 minutes. After that, BSC and DMAP were added and the mixture was stirred for an additional 2 hours. A halotag ligand was added, and the mixture was stirred for an additional 1 hour. Ethyl acetate was added to the reaction solution, and the mixture was washed with saturated brine. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by PTLC to give compounds 6b and 8b in yields of 23% and 45%, respectively.

[0077] Compound 6b: 1 H NMR(500 MHz, CDCl3)δ(ppm):. 13 C NMR(126 MHz)δ(ppm):HRMS(ESI,Positive-mode):m / z calcd for [C32H39ClN6O7+Na]+:677.2461,found 677.2472.

[0078] Compound 8b: 1 H NMR (500 MHz, CDCl) δ (ppm): 13 C NMR(126 MHz)δ(ppm):HRMS(ESI,Positive-mode):m / z calcd for [C36H49ClN6O7Si+Na]+:763.3013,found 763.3031.

[0079] Test 2. Synthesis of compounds (Figure 2) Hereinafter, we will specifically describe the synthesis methods for each compound shown in Figure 2. However, among the following compounds 9'-13', compounds 9', 10', and 12' are known compounds, so the synthesis methods will be omitted. [ka]

[0080] 2-1-1. Compound 11':7-(diethylamino)-4-(1-hydroxy-2-(trimethylsilyl)ethyl)-2H-chromen-2-one [ka]

[0081] Under an Ar atmosphere, a ((trimethylsilyl)methyl)magnesium chloride THF solution (1.0 M, 1.0 mL) was added dropwise to a 7-(diethylamino)-2-oxo-2H-chromene-4-carbaldehyde (92.8 mg) THF solution (10 mL). After stirring overnight at temperatures ranging from -78 °C to room temperature, the reaction was quenched by adding saturated aqueous ammonium chloride. The reaction solution was extracted three times with ethyl acetate, dried over magnesium sulfate, and concentrated. The residue was purified by column chromatography (conditions) to obtain 53.6 mg of the desired product in 42% yield.

[0082] 1 H NMR(500 MHz, CDCl3)δ(ppm):0.00(s, 9H),1.03(m,2H),1.08(t, 6H, 3J = 7.2 Hz), 3.28(q,4H,3J =7.2 Hz),4.97(m, 1H), 6.12(s,1H),6.42(br,1H),6.52(br,1H),7.34(d, 1H, 3J = 9.0 Hz). 13 C NMR(126 MHz)δ(ppm):0.7,12.4,26.6,45.6,68.5,99.3,105.0,107.4,109.6,125.5,149.5,156.0,161.0,162.8.

[0083] 2-1-2. Compound 11:1-(7-(diethylamino)-2-oxo-2H-chromen-4-yl)-2-(trimethylsilyl)ethyl benzylcarbamate [ka]

[0084] 7-(diethylamino)-4-(1-hydroxy-2-(trimethylsilyl)ethyl)-2H-chromen-2-one (39.0 mg) was dissolved in toluene (4.0 mL), diisopropylethylamine (20 μL) was added, and the mixture was stirred at room temperature for 5 minutes. Benzyl isocyanate (28 μL) was added, and the mixture was stirred at 115 °C overnight. Ethyl acetate was added to the reaction solution cooled to room temperature, washed with saturated brine, dried over magnesium sulfate, and concentrated. The residue was purified by column chromatography (conditions) to obtain 32.3 mg of the desired product in a 59% yield.

[0085] 1 H NMR(500 MHz, CDCl3)δ(ppm):0.08(s, 9H),1.17-1.26(m,8H),3.28(q, 4H, 3J = 7.1 Hz), 4.36(m,2H),5.23(br,1H),6.03(t, 1H, 7.4 Hz), 6.11(s,1H),6.57(br,1H),6.67(br,1H),7.25-7.37(m,5H),7.44(d, 1H, 3J = 9.1 Hz). 13 C NMR(126 MHz)δ(ppm):-0.9,12.5,24.6,45.3,45.4,70.2,99.1,104.9,109.6,125. 2,127.4,127.6,127.7,128.9,138.3,150.0,155.3,156.7,157.8,162.4.

[0086] 2-2-1. Compound 13':7-(diethylamino)-4-(1-hydroxybut-2-yn-1-yl)-2H-chromen-2-one [ka]

[0087] Under an Ar atmosphere, a THF solution of prop-1-yn-1-ylmagnesium bromide was added dropwise to a THF solution (10 mL) of 7-(diethylamino)-2-oxo-2H-chromene-4-carbaldehyde (64.8 mg). After stirring at -78 °C for 2 hours, the mixture was returned to room temperature and saturated aqueous ammonium chloride was added to quench the reaction. The reaction solution was extracted three times with ethyl acetate, dried over magnesium sulfate, and concentrated. The residue was purified by column chromatography (conditions) to obtain the desired product in 59% yield.

[0088] 1 H NMR(500 MHz, CDCl3)δ(ppm):1.18(t, 6H, 3J = 7.1 Hz), 1.85(d,3H,4J =2.1 Hz),3.19(br,1H),3.39(q, 4H, 3J = 7.1 Hz), 5.56(s,1H),6.38(s, 1H), 6.46(s,1H),6.57(d, 1H, 3J = 8.7 Hz), 7.63(d,1H,3J =8.7

[0089] 2-2-2. Compound 13:1-(7-(diethylamino)-2-oxo-2H-chromen-4-yl)but-2-yn-1-yl benzylcarbamate [ka]

[0090] 7-(diethylamino)-4-(1-hydroxybut-2-yn-1-yl)-2H-chromen-2-one was dissolved in dichloroethane (9 mL), diisopropylethylamine (13 μL) was added, and the mixture was stirred for 10 minutes. Benzyl isocyanate (18 μL) was added, and the mixture was heated to 85°C and stirred for 22 hours. Dichloromethane was added to the reaction solution, and the mixture was washed with saturated brine. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by column chromatography to obtain the desired product in 57% yield.

[0091] 1H NMR(500 MHz, CDCl3)δ(ppm):1.21(t, 6H, 3J = 7.0 Hz), 1.88(s, 3H), 3.41(q, 4H, 3J = 7.0 Hz), 4.41(d, 2H, 3J = 5.6 Hz), 5.21(t, 1H, 5.4 Hz), 6.32(s, 1H), 6.51(s, 1H), 6.53-6.60(m, 2H), 7.23-7.35(m, 5H), 7.53(d, 1H, 3J = 9.1 Hz). 13 C NMR(126 MHz)δ(ppm):4.0, 12.6, 44.9, 45.5, 62.7, 74.1, 85.5, 97.9, 105.9, 107.2, 108.8, 125.7, 127.7, 128.9, 137.9, 150.7, 150.8, 154.8, 156.7, 162.2.

[0092] 2-3. Compound 9, Compound 10, Compound 12 The synthesis of Compounds 9, 10, and 12 was carried out in the same manner except for the coumarin substrate. The coumarin substrate was dissolved in a solvent, and two equivalents of diisopropylethylamine were added. The mixture was stirred at room temperature for 5-10 minutes. Two equivalents of benzyl isocyanate were then added, and the mixture was heated and stirred overnight. The reaction solution was washed with saturated saline, and the organic layer was dried over magnesium sulfate and concentrated. The residue was purified by column chromatography to obtain the target product.

[0093] Compound 9: 1H NMR(500 MHz, CDCl3)δ(ppm):1.21(t,6H,7.0 Hz),3.42(q, 4H, 7.0 Hz), 4.42(d,2H,3J =6.0 Hz),5.26(s, 2H), 5.33(br,1H),6.18(s, 1H), 6.63(br,1H),6.73(br,1H),7.27-7.37(m,6H). 13 C NMR(126 MHz)δ(ppm):12.3,45.4,46.0,62.0,107.4,124.7,127.6,127.7,127.8,128.8,128.9,138.1,150.2,155.6,156.1,161.7.

[0094] Compound 10: 1 H NMR(500 MHz, CDCl3)δ(ppm): 1.21(t, 6H, 7.1 Hz), 1.57(d, 3H, 3J = 6.7 Hz), 3.41(q, 4H, 3J = 7.1 Hz), 4.33 - 4.42(m, 2H), 5.23(t, 1H, 3J = 5.4 Hz), 6.04(q, 1H, 3J = 6.7 Hz), 6.15(s, 1H), 6.52(d, 1H, 4J = 2.3 Hz), 6.61(d, 1H, 3J = 8.9 Hz), 7.28 - 7.36(m, 5H), 7.42(d, 1H, 3J = 8.9 Hz). 13 C NMR(126 MHz)δ(ppm): 12.6, 21.2, 45.0, 45.4, 68.0, 98.4, 105.0, 106.2, 109.0, 125.0, 127.7, 127.8, 128.9, 138.2, 150.6, 155.4, 156.3, 156.7, 162.4.<s

[0095] Compound 12: 1 H NMR(500 MHz, CDCl3)δ(ppm): 1.19(t, 6H, 7.0 Hz), 1.77(s, 3H), 1.94(s, 3H), 3.40(q, 4H, 3J = 7.0 Hz), 4.37(d, 2H, 3J = 6.0 Hz), 5.20(t, 1H, 3J = 5.4 Hz), 5.27(d, 1H, 3J = 9.2 Hz), 6.17(s, 1H), 6.49(d, 1H, 4J = 2.5 Hz), 6.55(d, 1H, 3J = 9.1 Hz), 6.59(d, 1H, 3J = 9.2 Hz), 7.26 - 7.39(m, 5H). 13 C NMR(126 MHz)δ(ppm): 12.5, 18.8, 25.8, 44.7, 45.3, 69.1, 98.0, 105.6, 106.3, 108.6, 121.4, 125.4, 127.5, 127.6, 128.7, 138.1, 140.7, 150.4, 154.7, 155.3, 156.6, 162.3.

[0096] Test 3. Photocleavage test 1 In this test, the photocleavage activity of compounds (9-13) synthesized in Test 2 was evaluated. 1 Quantitative analysis was performed using H NMR measurement. Specifically, each compound was first dissolved at approximately 10 mM in d6-DMSO containing 1% DO to prepare a measurement sample. Using an LED light (ASAHI SPECTRA, CL-1503), the measurement sample in a quartz vial was irradiated with light at an intensity of 14 mW and a wavelength of 405 nm. 1 The H NMR signal was followed over time and the results are shown in Figure 3a.

[0097] The upper part of Figure 3a shows the reaction of compound 11 cleaved by light irradiation. 1 This shows the change over time in H NMR signals due to light irradiation. With the passage of time, the proton signals indicated by * in Figure 3a weakened, while the proton signals indicated by ** strengthened.

[0098] It also shows a significant change 1 The photocleavage activity was quantified from the integrated value of the H signal 30 minutes after the start of light irradiation. The results are shown in Figure 3b. Compounds 11 and 12 were confirmed to have higher photocleavage activity.

[0099] Test 4. Conjugation of Compounds to Proteins and Immobilization on Solid Phases Compounds (6a, 6b, 8a, 8b) synthesized in Test 1 (10 mM) were mixed with commercially available biotinylated azide compounds (10 mM) at a concentration ratio of 3:1 and reacted at 60°C overnight to quantitatively biotinylate compounds (6a, 6b, 8a, 8b) (compounds (6a', 6b', 8a', 8b')).

[0100] Compounds 6a', 6b', 8a', and 8b' each possess a ligand moiety with high binding affinity to the HaloTag protein, allowing conjugates to be obtained simply by mixing them in solution. Taking advantage of this property, we performed chemical ligation reactions between compounds 6a', 6b', 8a', and 8b' and HaloTag-fused NanoLuc protein. Specifically, a DMSO solution of compounds 6a', 6b', 8a', and 8b' was mixed with an aqueous solution of HaloTag-fused NanoLuc in a buffer solution (50 mM Tris-HCl, 100 mM NaCl, pH 7.5) and incubated on ice for 1 hour. Three equivalents of HaloTag-fused NanoLuc were added to each compound 6a', 6b', 8a', and 8b', and the DMSO concentration in the final solution was adjusted to less than 1%. The ligation reaction was confirmed by electrophoresis. Fluorescence from compounds (6a', 6b', 8a', 8b') was confirmed in a band of molecular weight size equivalent to that of HaloTag-fused NanoLuc. The resulting solution contained the target compounds (6a", 6b", 8a", 8b"), in which NanoLuc was linked to biotin via a photocleavable structure, as well as unreacted HaloTag-fused NanoLuc and the compounds (6a, 6b, 8a, 8b) that had been added in excess in the previous reaction. Since only biotinylated molecules bind to the solid phase in the subsequent immobilization reaction, the resulting reaction solution was used directly in the subsequent solid-phase immobilization reaction.

[0101] Next, compounds 6a, 6b, 8a, and 8b were immobilized onto magnetic beads. Streptavidin-immobilized magnetic beads (Dynabeads MyOne Streptavidin C1) were dispersed in an aqueous solution containing compounds 6a, 6b, 8a, and 8b, and the solution was left to stand on ice for 1 hour. The magnetic beads were then precipitated using a magnet, and the supernatant was removed. The solution was then replaced with an aqueous buffer solution (50 mM Tris-HCl, 100 mM NaCl, pH 7.5) and washed three times. The washed magnetic beads carrying compounds 6a, 6b, 8a, and 8b were dispersed in an aqueous buffer solution (50 mM Tris-HCl, 100 mM NaCl, pH 7.5) and used in the photocleavage test (Test 5) and hydrolysis test (Test 6), which will be described later.

[0102] A schematic diagram of (8a")-loaded magnetic beads is shown in Figure 4. The upper part of Figure 4 shows how the portion containing HaloTag-fused NanoLuc is separated from the (8a")-loaded magnetic beads by photocleavage or hydrolysis. The lower part of Figure 4 shows the state in which compound (8a") has been immobilized on the magnetic bead solid phase.

[0103] Test 5. Photocleavage test 2 To investigate the photocleavage efficiency of compounds (6a, 6b, 8a, and 8b), we quantitatively analyzed the photoirradiation time dependence of the amount of protein released from the solid phase using magnetic beads bearing (6a", 6b", 8a", and 8b"). Aqueous dispersions of (6a", 6b", 8a", and 8b") bearing magnetic beads were irradiated with light at 405 nm and an intensity of 8.4 mW for periods ranging from a few seconds to 3 minutes. After irradiation, the magnetic beads were precipitated using a magnet, and the supernatant was removed. Furimazine, a luminescent substrate for NanoLuc, was added to the removed supernatant, and the luminescence intensity was measured after 1 minute. In other words, in this test system, the higher the photocleavage activity, the greater the amount of protein released from the solid phase and present in the supernatant, resulting in stronger luminescence.

[0104] The results are shown in Figure 5. The vertical axis on the left of Figure 5 represents luminescence intensity, and the horizontal axis represents light irradiation time (seconds). The center of Figure 5 shows the structures of the parts involved in photocleavage and hydrolysis of compounds (6a", 6b", 8a", and 8b") (the structures of other parts are omitted). The right of Figure 5 shows the luminescence intensity of each evaluation sample after 3 minutes of light irradiation. It was confirmed that the ester derivative of compound 8a" bearing a trimethylsilyl group was the most efficient at photocleavage and cleaving the protein. Furthermore, in a comparison between compounds 6b" and 8b", it was confirmed that compound 8b" bearing a trimethylsilyl group was the more efficient at photocleavage.

[0105] Test 6. Hydrolysis test 6-1. To examine the hydrolysis resistance of compounds (6a, 6b, 8a, 8b), magnetic beads carrying (6a", 6b", 8a", and 8b") were dispersed in buffers of pH 5.3, 7.3, and 9.1 and incubated at 37°C for 30 minutes. The magnetic beads were then precipitated using a magnet, and the separated supernatant was mixed with furimazine, a luminescent substrate for NanoLuc, and the luminescence intensity was measured after 1 minute. In other words, in this test system, the higher the hydrolysis resistance, the less protein present in the supernatant and the weaker the detected luminescence intensity.

[0106] The results are shown in Figure 6. As is clear from the comparison between 8b" and 6b", and between 8a" and 6a", the introduction of trimethylsilyl groups improved hydrolysis resistance.

[0107] 6-2. In addition, esterase, an ester bond-degrading enzyme, was mixed with an aqueous dispersion of magnetic beads (6a”, 6b”, 8a”, 8b”) and incubated at 37°C for 25 minutes. The magnetic beads were then precipitated using a magnet, and the separated supernatant was mixed with furimazine, a luminescent substrate for NanoLuc, and the luminescence intensity was measured after 1 minute.

[0108] The results are shown in Figure 7. As is clear from the comparison between 8b" and 6b", and between 8a" and 6a", the introduction of a trimethylsilyl group improved hydrolysis resistance even in the system in which esterase was added.

Claims

1. General formula (I): 【Chemistry 1】 (In the formula, R １ａ , R １ｂ , and R １ｃ are each independently any hydrocarbon group; R ２ is any group containing a fluorescent moiety; X is NH or CH ２ is; Y １ is any group) or a salt thereof, Upon irradiation with light, a compound represented by general formula (II): 【Chemistry 2】 (In the formula, R １ａ , R １ｂ , R １ｃ , and R ２ is the same as in general formula (I). A composition for producing a compound represented by the formula:

2. In general formula (I), the R ２ is represented by the general formula (III): 【Transformation 3】 (In the formula, R ３ is any electron donating group; R ４ａ , R ４ｂ , R ４ｃ , and R ４ｄ are each independently a hydrogen atom or any group. The composition of claim 1 ,

3. In general formula (I), R １ａ , R １ｂ , and R １ｃ The composition according to claim 1 or 2, wherein each of the is independently an alkyl group having 1 to 5 carbon atoms or a phenyl group.

4. 3. The composition according to claim 1, wherein the phosphor portion absorbs light having a wavelength of 350 nm to 850 nm, and the light irradiation is light having a wavelength of 350 nm to 850 nm.

5. General formula (I): 【Chemistry 4】 (In the formula, R １ａ , R １ｂ , and R １ｃ are each independently any hydrocarbon group; R ２ is any group containing a fluorescent moiety; X is NH or CH ２ is; Y １ is any group) or a salt thereof, General formula (II): 【Transformation 5】 (In the formula, R １ａ , R １ｂ , R １ｃ , and R ２ is the same as in general formula (I). A method for obtaining a compound represented by the formula:

Images